ARF suppression by MYC but not MYCN confers increased malignancy of aggressive pediatric brain tumors

Medulloblastoma, the most common malignant pediatric brain tumor, often harbors MYC amplifications. Compared to high-grade gliomas, MYC-amplified medulloblastomas often show increased photoreceptor activity and arise in the presence of a functional ARF/p53 suppressor pathway. Here, we generate an immunocompetent transgenic mouse model with regulatable MYC that develop clonal tumors that molecularly resemble photoreceptor-positive Group 3 medulloblastoma. Compared to MYCN-expressing brain tumors driven from the same promoter, pronounced ARF silencing is present in our MYC-expressing model and in human medulloblastoma. While partial Arf suppression causes increased malignancy in MYCN-expressing tumors, complete Arf depletion promotes photoreceptor-negative high-grade glioma formation. Computational models and clinical data further identify drugs targeting MYC-driven tumors with a suppressed but functional ARF pathway. We show that the HSP90 inhibitor, Onalespib, significantly targets MYC-driven but not MYCN-driven tumors in an ARF-dependent manner. The treatment increases cell death in synergy with cisplatin and demonstrates potential for targeting MYC-driven medulloblastoma.

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Software and code
Policy information about availability of computer code Data collection RNA-seq: Raw RNA-seq reads were mapped to the mm9 mouse genome via the STAR alignment algorithm (Version 2.7.2b) and the BOWTIE2 alignment algorithm (Version 2.3.4.3). Gene specific read counts were obtained via the featureCounts function from the Subread package (version 1.5.2). Exome Seq: Mouse tumor biopsies, normal control sample pairs and GMYC1 and GTML2 tumor cell lines were whole exome sequenced by the National Genomics Infrastructure SNP&SEQ Technology Platform (Uppsala). Sequencing library preparation were performed using Twist Mouse Exome Panel Kit (Twist Bioscience). Clustering generation and paired-end sequencing were run for 100 cycles in one flowcell using the NovaSeq 6000 system (Illumina). A detailed analysis of specific mutations and allele frequencies on the Trp53 transcript (ENSMUST00000108658.9) on Chr.11 was performed. Methylation Arrays: The methylation in DNA from mouse tumor samples was profiled using the MM285 Infinium MouseMethylation BeadChip (Illumina).
Data analysis RNA-seq: Differential expression analyses were conducted in R using the edgeR (Version 3.28.1) package. Batch-effect removal was performed via the limma (Version 3.42.2) package. Mouse to Human Ortholog mappings/translation was conducted through the biomaRt (Version 2.42.1) package. The metagene R scripts (Tamayo et al. (2007); https://doi.org/10.1073/pnas.0701068104) was used for cross-species analyses. Exome Seq: Reads were aligned to the GRCm38.p6 reference genome build using Burrows-Wheeler Aligner version 0.7.17. Bam files were converted using Samtools v. 1.14 and duplicated reads where marked using Picard v. 2.23.4 (https://broadinstitute.github.io/picard/). Variants in tumor-normal and tumor-only samples were called with VarScan v. 2.3.9 using somatic and mpileup2cnettings respectively. Somatic variants from tumor-only samples were obtained by excluding all variants reported in the normal samples. All samples were annotated using SnpEff v. 5.0C. Methylation Arrays: Pre-processing of beta values from human samples were performed using the minfi package (v1.24.0) and the IlluminaHumanMethylation450kmanifest (v0.4.0) package. Differential methylation analyses, including significance testing, of CDKN2A specific

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CpG probes between MB samples were performed using the dmpFinder function from the minfi package. Following raw data generation of mouse samples, all IDAT files were processed in R (v4.1.2) using the package SeSAMe (version 1.14.2). and annotated with the MM285 Infinium Mouse Methylation Manifest 12v1-0 manifest. Using the manifest, probes known to be poor quality (e.g., cross-hybridizing, SNP-enriched) were masked and remaining data values normalized using normal-exponential out-of-band (noob) method (Triche et al. (2013); https://doi.org/10.1093/nar/gkt090). Animal survival was graphically shown as a Kaplan-Meier curve, made and assessed using GraphPad Prism 8 or 9 software.
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Data
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Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.

Reporting on sex and gender
The study is not involving human research participants.

Population characteristics
Population characteristics from publically available data mostly includes primary biopsies from tumors in children (under 18 years old) with no selection on a specific sex.

Recruitment
No participants were recruited to this study. We only used publically available data on patient collections where a specific brain tumor was identified upon diagnosis.

Ethics oversight
We used publically available data on patient collections where a specific brain tumor was identified.
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Life sciences study design
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Sample size
Sample sizes for in vivo experiments, e.g. treatment cohorts, were used on the basis of agreeing to a size suitable for ethical regulations while also large enough to ensure strong statistical power. Sample sizes were large enough to ensure results obtained were of a representable quantity and quality. For global expression analysis more than 3 samples of each group was usually included. For in vitro experiments, initial samples size for the number of new cell lines generated (GMYC model) was n=3. We also used at least three different lines for dox treatments, DNMT inhibition or HSP90 inhibition. When direct comparisons were used for more precise follow up comparisons -a representative cell line could be directly compared against another representative line. Such experiments were always confirmed and repeated at least three times. When e.g. protein measurements or histological analyses, numerous biological repeats were conducted to ensure a representative overview.
Data exclusions A SHH MB was excluded from the GTML tumors collected (from GSE162080) and analyzed as it was not designated to the correct human subgroup (Group 3 MB) based on global expression profiling. The rationale for this exclusion has also been described in the manuscript text. For survival analysis of animals that were found dead without obvious signs of tumor (primary tumor penetrance studies) they were marked as censored as described in Methods.

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Finally, to avoid irrelevant survival effects caused by secondary tumors that might arise in long-term survivors, patients with survival times longer than 10 years (according to publically available clinical data) were censored at exactly 10 years as described in Methods.
No other data has been excluded from this study.

Replication
For applicable situations, experiments were replicated numerous times (>3). Analysis of animal tissue was carried out for many animals across multiple generations to ensure no genetic changes occured and that all data shown was an accurate and average representation of these samples. Where appropriate, statistical analysis has been carried out for these replicates. The work we present is reliable as it is a combination of many findings, both confirmed in vivo and in vitro. For ethical reasons some animal experiments were not repeated. Instead enough animals were analyzed in order to prove statistically significant differences.
Randomization Before graft injections, mice injected were randomized into different treatment arms/cages. Animal treatment groups were not randomised.
Genotyping was carried out to ensure all mice contained the correct transgenes (GMYC: Glt1-tTA, TRE-MYC and GMYC/ARF model: Glt-tTA-TRE-MYC-ARF fl/fl). Once all mice were confirmed to contain the correct transgenes, cohorts were divided without randomisation. All experiments involving intracranial injections used the same strict injection coordinates and numbers of cells injected, as all injections were carried out by one researcher, to eliminate any bias.

Blinding
Blinding was performed where the responsble researcher blinded results that were analysed/quantified without knowing the name of the investigated sample. In vivo work and mouse symptoms were assessed by multiple researchers as well as the technical staff in the animal facility. Unless stated otherwise, all mice were sacrificed at a humane endpoint judged to be similar across all individual animals.

Validation
Antibodies were used as per the manufacturers' instructions. For important stainings, antibodies to discover the same protein a comparative analysis was performed.

Eukaryotic cell lines Policy information about cell lines and Sex and Gender in Research
Cell line source(s) GMYC1, GMYC2, GMYC3 cell lines were derived from tumors of the transgenic mice generated in this study. GTML2 were derived from a transgenic mouse derived in previously published studies (Swartling et al., Genes & Dev, 2010). GMYC1/+MYC and GTML2/+MYC cell lines were generated from the GMYC1 and GTML2 cell lines previously mentioned, using lentivirally overexpressing MYC, carried out in this study.

Authentication
None of the mouse cell lines used were authenticated as they were directly maintained in low passage numbers after generation. Human lines were analyzed with STR profiling by Sigma Aldrich and ATCC (before ordering from them), to confirm correct donor our species.

Mycoplasma contamination
Cell lines used were contamination-free when obtained from repositories or vendors but tested regularly upon culturing and passaging using a Mycoplasma detection kit (MycoAlert) from Lonza. Only confirmed mycoplasma-free lines were used in research.